To establish the basis for the reduced expression of the X determinant on leukemic blasts and the changes in antigenic expression that occur during myeloid maturation, the presence on myeloid cells of X and related structures was examined in conjunction with studies on the activities of the glycosyltransferases involved in their biosynthesis. Expression of X and sialyl-X was weak on blasts in comparison with neutrophils despite the presence of the requisite precursor structures. Much higher levels of 3-fucosyltransferase activity were found in blasts than in neutrophils when nonsialylated substrates were used, but, whereas the enzyme in neutrophils reacted equally well with 3′- sialylated and nonsialylated acceptors, the enzyme in blasts showed a marked preference for nonsialylated substrates. 6′-Sialyltransferase activity was strong in blasts but was not detectable in neutrophils, whereas a much lower level of 3′-sialyltransferase activity was present in both blasts and neutrophils. Dimethyl sulfoxide-induced maturation of HL60 cells was associated with (1) a decrease in both 6′- sialyltransferase and 3-fucosyltransferase activities, (2) a change in the substrate specificity of 3-fucosyltransferase towards that found in mature cells, and (3) increased cell surface expression of sialyl-X. These results suggest that the reduced expression of X in myeloblasts is related to the presence of the strong 6′-sialyltransferase, which uses the precursor substrate at the expense of the 3-fucosyltransferase and prevents the synthesis of X and sialyl-X. The developmental regulation of the levels of 3′- and 6′-sialyltransferases, and the level and specificity of the 3-fucosyltransferases, therefore controls the expression of X and its degree of sialylation.